Tyrosine kinase inhibitors, such as Imatinib, are currently used as effective and frontline therapy for chronic-phase of chronic myeloid leukemia (CML). However, despite the generally positive response to the clinical therapy, the resistance to Imatinib represents a serious problem in the treatment of patients expressing the Bcr-Abl fusion gene, resulting from the juxtaposition of the c-Abl proto-oncogene on chromosome 9 to Bcr sequences on chromosome 22. The identification of Imatinib-resistant Bcr-Abl mutations has led to a rapid development of new generations of Bcr-Abl inhibitors with distinct mechanisms of action (Leukemia, 2008, 22, 572-577). The clinical activity of these new derivatives, such as AMN107 and SKI606, is currently evaluated in ongoing Phase I/II clinical trials, while Dasatinib, a dual Src/Abl inhibitor, has received FDA approval for clinical treatment of Imatinib-resistant CML patients (Cancer Cell. 2005, 7, 129-141; Curr Pharm Biotechnol. 2006, 7, 371-379; Clin Ther. 2007, 29, 2289-2308). However, although these agents are in general very active in treating Imatinib-resistant CML, they fail to overcome the Imatinib resistance caused by the T315I mutation. This mutation, occurring at the gatekeeper position of the Abl kinase domain, is responsible for about 15% of Imatinib-resistant CML patients and it is considered the biggest obstacle for CML treatment, especially in advanced phases of the disease. In the last few years, multiple efforts have been focused on the development of new Bcr-Abl inhibitors targeting this mutant. Recently, two Aurora kinase inhibitors, VX-680 (Cancer Res. 2006, 66, 1007-1014) and PHA-739358 (Nat. Rev. Cancer 2007, 7, 345-356) and the new compound PPY-A (Chem. Biol. Drug Des. 2007, 70, 171-181) have been reported as effective against the deleterious mutations at the gatekeeper position. Since their discovery, many other novel agents have been developed, i.e., SGX393 (Proc. Natl. Acad. Sci. USA. 2008, 105, 5507-5512).
To attenuate Bcr-Abl transforming potential and overcome drug resistance, much interest is addressed toward development of new and complementary therapeutic strategies targeting pathways directly involved in the regulation of causative events of CML. In this context, the authors of the present invention focused their efforts on studying 14-3-3 proteins and their relationship with CML. 14-3-3s were the first proteins described as phosphoserine or phosphothreonine binding modules (Seminars in Cancer Biology 2006, 16, 173-182). In human, seven distinct isoforms have been identified (β, γ, ε, η, σ, τ, and ζ, corresponding to the entries P31946, P68981, P62258, Q04917, P31947, P27348, and P29310 of the UniProtKB/Swiss-Prot database, respectively) and they constitute a family of highly conserved and ubiquitously expressed proteins, acting as homo or heterodimers. The initial observation that the binding to 14-3-3 family members requires ligand phosphorylation emerged from work on tryptophan hydroxylase (Biochem. Biophys. Res. Commun. 1993, 194, 144-149) and Raf, the upstream activator of the classical MAP kinase pathway (Mol. Cell. Biol. 1995, 15, 3390-3397). Subsequent studies of the 14-3-3 binding sites on Raf (Cell. 1996, 84, 889-897), together with oriented peptide library screening on all mammalian 14-3-3s (Cell. 1997, 91, 961-971), led to the identification of two main 14-3-3 consensus motifs. These motifs correspond to the sequences RSX-pS/T-XP (mode I) and RXXX-pS/T-XP (mode II), where pS/T denotes a phosphorylated Serine or Threonine residue and X any amino acid, and they recognized by all 14-3-3 isotypes. 14-3-3s bind a large number of protein targets involved in the regulation of many intracellular processes, such as cell cycle progression, protein trafficking, signal transduction, cytoskeletal rearrangements, metabolism, transcriptional regulation of gene expression. 14-3-3s play also an important role in the coordination and the regulation of DNA damage response and apoptosis (Cell Cycle 2005, 4, 777-779; Seminars in Cancer Biology 2006, 16 162-172; Leukemia 2008, 22, 572-577). In this regard, c-Abl plays an important intermediary role in inducing apoptosis cell death (Nature Cell Biology 2005, 7, 213-214; Nature Cell Biol. 2005, 7, 278-285; Cell Cycle 2005, 4, 777-779; EMBO J. 2006, 25, 3774-3783). In fact, normally, c-Abl can shuttle between the cytoplasm and nucleus by classical mechanisms referring to the presence of three nuclear localization signals (NLSs) and one nuclear export signal (NES) in c-Abl carboxy-terminal region (entry P00519 of the UniProtKB/Swiss-Prot database). Into the nucleus, c-Abl can induce apoptosis in response to DNA damage, demonstrating that its intracellular localization is a crucial aspect in causing either the survival or apoptosis of the cell. In normal cells, the cytoplasmatic localization of c-Abl is due to the binding with 14-3-3 proteins. In fact, in the sequence of c-Abl is present a consensus motif for this protein family, located between the second (residues 707-720 of Abl sequence) and the third (residues 759-772 of Abl sequence) NLS, corresponding to the sequence RSV-T(735)-LP (entry P00519 of the UniProtKB/Swiss-Prot database). Mutagenesis studies on Thr735 of Abl revealed that this sequence, after phosphorylation on this threonine residue, allows the binding to 14-3-3s in such a way that c-Abl is sequestered into the cytoplasm (Nature Cell Biol. 2005, 7, 278-285). Upon DNA damage, a mechanism involving c-Jun N-terminal kinase (Jnk) is activated in order to phosphorylate 14-3-3s on specific serine residues, inducing both c-Abl release from 14-3-3s and its localization into the nucleus to activate apoptosis cell death (Nature Cell Biol. 2005, 7, 278-285; Nature Cell Biol. 2005, 7, 213-214).
The oncogenic form of Abl kinase, Bcr-Abl, is constitutively activated and localizes primarily to the cytoplasm (J. Clin. Invest. 1993, 92, 1925-1939) where it elicits anti-apoptotic signals and confers survival. With regard to the mechanism of association and dissociation between c-Abl and 14-3-3, the fusion protein Bcr-Abl interferes in many ways. First of all, Bcr-Abl prevents the translocation of c-Abl into the nucleus in response to ionizing radiations by inhibiting the phosphorylation by Jnk of 14-3-3σ at Ser186. In CML cells, in fact, it was demonstrated that Jnk kinase binds to the Bcr-Abl/HDAC1 complex more efficiently than to the complex c-Abl/14-3-3σ, precluding or reducing its ability to phosphorylate 14-3-3σ and to release c-Abl. In addition, Bcr-Abl affects the complete activation of Jnk preventing its phosphorylation at Thr183, indispensable for the subsequent phosphorylation of 14-3-3. Experimental evidences indicate that inhibition of the fusion protein enzymatic activity by the inhibitor Imatinib is followed by phosphorylation of 14-3-3σ at Ser186 and of Jnk at Thr183, resulting in the translocation of c-Abl into the nuclear compartment. Secondly, Bcr-Abl in CML cells induces a massive over-expression of 14-3-3σ through an epigenetic regulation (hyper-acetylation of histone H4) of 14-3-3 promoter. R18 is a peptide inhibitor of 14-3-3 (Biochemistry, 1999, 38, 12499-12504). Once this peptide is bound to the 14-3-3 binding site, proteins with a consensus motif for 14-3-3 can not interact with 14-3-3s. In this context, R18, bound to 14-3-3 binding site, prevents the binding of c-Abl that can be, in that way, translocated into the nucleus to induce apoptosis. Moreover, with regard to the previously described effect of Bcr-Abl on the over-expression of 14-3-3σ, the inhibition of Bcr-Abl mediated by Imatinib resulted in persistent deacetylation of histone H4 at 14-3-3σ promoter, with a significant reduction in the amount of 14-3-3σ protein expression. This aspect can facilitate the translocation into the nucleus of the c-Abl protein (Traffic, 2009, 10, 637-647).
All together these results suggest that i) targeting the 14-3-3σ binding site with molecules able to affect the interactions with the protein target, and with Abl in particular, and ii) simultaneously inhibiting Bcr-Abl oncoprotein, can represent an alternative and/or complementary therapeutic strategy to treat CML.
At the present time, only R18 is used as an inhibitor of 14-3-3 proteins to block the binding between 14-3-3s and their target. In the present invention, computational methodologies were applied to identify non peptidic compounds able to disrupt the interaction between 14-3-3s and their protein ligands, and in particular, between 14-3-3σ and c-Abl. The biological effects of the identified compounds were tested in cellular assays in order to verify their mechanism of action. In particular, clonogenic assays, evaluations of apoptotic cell death and measurements of nuclear translocation of c-Abl were performed.